PUFAs are polyunsaturated fatty acids with a long hydrocarbon chain composed of 18 or more carbon atoms having two or more double bonds and a terminal carboxylate group.
The properties of polyunsaturated fatty acids are highly influenced by the position of the double bond, and one differentiates omega-3 PUFAs, which have the first double bond at the third position counting from the methyl end of the carbon chain, and omega-6 PUFAs, which have the first double bond at the sixth position counting from the methyl end of the carbon chain. Eicosapentaenoic acid belongs to the former group, particularly eicosapentaenoic acid with double bonds in position 5, 8, 11, 14 and 17 (EPA) and docosahexaenoic acid, particularly docosahexaenoic acid with double bonds in position 4, 7, 10, 13, 16, 19 (DHA), while, for example, arachidonic acid (ARA) belongs to the latter group.
PUFAs are essential for humans, and it has been proven that they have many beneficial effects on human health, including proper development of brain and visual functions and prevention of disease, such as cardiovascular disease and cancer.
The omega-6 PUFA arachidonic acid plays an important role in the structure and function of biological membranes, and is a precursor of the biologically active prostaglandins and leukotrienes. Arachidonic acid is necessary for the neurological and neurophysiological development of both term and preterm infants, and many expert organizations, including the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) recommend that infant formula should be supplemented with arachidonic acid.
The omega-3 PUFA EPA and DHA, as well, possess a number of physiological functions in humans. They are part of the human tissue, and in the rod outer segment in the retina, DHA represents more than 60% of the total fatty acids. DHA is regarded to be essential for proper visual function and neurological development of infants. Preterm and young infants are unable to synthesize sufficient amounts of DHA and receive the remaining by breast milk. DHA also reduces or eliminates the risk factor involved in various diseases like cardiovascular diseases and exerts positive effects on hypertension, arthritis, arteriosclerosis and thrombosis.
Various publications, patents and patent application focus on the production of PUFA from fatty acid substrates. Recently, the pathway from linoleic acid to arachidonic acid was reconstituted in S. cerevisiae (Domergue et al., 2003, Beaudoin et al., 2000) and synthesis of polyunsaturated fatty acids was established by supplying the precursor metabolite, linoleic acid, to the medium.
Other groups have established parts of the PUFA pathway in reconstitution experiments. For example, U.S. Pat. No. 6,432,684 describes the sequence of a delta-5 desaturase from human, which, when expressed in yeast, produces arachidonic acid, when dihomo-gamma-linolenic acid is supplied.
U.S. 2002/0170090 describes an omega-3 desaturase from Caenorhabditis elegans and its expression in various organisms including bacteria, cyanobacteria, phytoplanton, algae, fungi, plants and animals, and the production of a lipid from an organism that expresses the omega-3 desaturase. The enzyme catalyzes the conversion of omega-6 fatty acids with 18, 20 and 22 carbon atoms to the corresponding omega-3 fatty acids. Yeast cells, expressing the omega-3 desaturase from C. elegans, converted exogenously supplied linoleic acid and omega-6 docosatetraenoic acid into alpha-linolenic acid and omega-3 docosapentaenoic acid, respectively.
PCT/US98/07422 describes the isolation of a delta-5 desaturase from Mortierella alpina and expression of said enzyme in microbial cells, particularly in Saccharomyces cerevisiae, and reports production of arachidonic acid when dihomo-gamma-linolenic acid is supplied in the growth medium. WO 99/27111 describes a delta-6 desaturase from C. elegans and its expression in yeast, which led to production of gamma-linolenic acid from exogenously, supplied oleic acid. In WO99/33958, the expression of a delta-5 desaturase (originally obtained from C. elegans) in microorganisms, such as algae, bacteria and fungi, and particularly, its expression in yeast is disclosed. WO 02/44320 describes a number of different human elongases, many of which have been tested for functionality in yeast using a number of different fatty acids as externally supplied substrates.
A method for the production of arachidonic acid in transgenic organisms (WO 03/012092) has been applied. Here, the inventors describe the expression of a delta-5 desaturase, which leads to the production of arachidonic acid in yeast; however, it requires dihomo-gamma linolenic acid as an external substrate.
The inventors of PCT/US98/07421 test the expression of various desaturases including delta-12 desaturase, delta-6 desaturase and delta-5 desaturase and reconstitute the function of these enzymes by adding fatty acid substrates to the growth medium and analysing their conversion.
In all the above-mentioned publications, patents and patent applications, processes have been described, where it is necessary to supply fatty acids as external substrates in the medium in order to produce PUFAs.
In the following a few publications are described, which report production of PUFAs with up to 3 double bonds from non-fatty acid substrates. In U.S. Pat. No. 6,355,861 it is shown that the expression of delta-12 and delta-6 desaturase from Cynecosystis in Cynecococcus leads both to the production of linoleic acid and gamma-linolenic acid, fatty acids with two double bonds and three double bounds, respectively. Furthermore, expression of a delta-6 desaturase from prime rose in a bacterial, fungal or plant cell is disclosed, including expression of said delta-6 desaturase in various plants for the production of gamma-linolenic acid.
U.S. Pat. No. 6,136,574 describe the production of gamma-linoleic acid in yeast from endogenously available oleic acid. PCT/US98/07126 describes the expression of a delta-6 desaturase and a delta-12 desaturase and reports, for example that expression of these genes in a host cell leads to the production of gamma-linoleic acid.
No prior art reference discloses successful heterologous PUFA production with four or more double bonds in microorganisms from carbon sources other than fatty acids, despite the fact that a high number of different genes involved in PUFA biosynthesis have been identified. This clearly demonstrates that it is a difficult task to produce PUFAs with four or more double bonds at sufficient or detectable titers in microorganisms that usually do not produce PUFA. Although the inventors of U.S. 2003/0177508 describe sequences of four genes that are involved in PUFA elongation and show the function of all these genes, the inventors can only speculate in an example (example III) that expression of delta-12 desaturase, delta-6 desaturase, delta-5 desaturase, and a Mortierella alpina elongase cDNA in yeast could result in the production of arachidonic acid without the need of exogenous supply of fatty acids.
As an intermediate summary of the above paragraphs, it can be concluded that, except for the speculations specified in U.S. 2003/0177508, there have until now been no reports on expressing a heterologous pathway for the production and PUFA from non-fatty acid substrates in microorganisms. Until now, reports concerning heterologous PUFA production from non-fatty acid substrates in microscopic hosts, such as yeast, have been limited to PUFAs with less than four double bonds, namely three or less double bonds, such as linoleic acid and gamma-linolenic acid.
If the strategy suggested in U.S. 2003/0177508 is followed, one will expect a low content of arachidonic acid in bakers yeast, as this organism has a low content (approximately 10% of cell dry weight) of fatty acids. Furthermore, the fatty acids in bakers yeast primarily consists of fatty acids with 16 carbon atoms, and the most dominant mono-unsaturated fatty acid is palmitoleic acid, which can not serve as a precursor for synthesis of arachidonic acid. The result of simply expressing the mentioned four genes in S. cerevisiae, where expression of these four genes, results in an arachidonic acid content of 0.8% of the fatty acids, or corresponding to less than 0.08% of the yeast dry weight.
The production of polyunsaturated omega-3 and omega-6 fatty acids with four and five double bonds, but not six double bonds, has recently been reported in plants. Qi and co-workers were able to produce the omega-3 fatty acid EPA and the omega-6 fatty acid arachidonic acid in the plant Arabidopsis thaliana (Qi et al. 2004).
Qi and co-workers show for the first time that arachidonic acid and EPA can be produced via a heterologous pathway in an organism, such as a plant, using a non-fatty acid substrate. The authors succeed by simultaneously expressing genes coding for delta-9 elongase, delta-8 desaturase and delta-5 desaturase, an approach that makes use of the endogenous delta-12 desaturase and endogenous omega-3 desaturase activities of A. thaliana for production of arachidonic acid and EPA. In many organisms, including microorganisms, such as many yeasts and filamentous fungi, it would be necessary to express at least 4 or at least 5 heterologous genes in order to produce PUFA with at least four or at least five double bonds. Until now the expression of more than 3 heterologous genes at the same time for the production of PUFAs has never been applied. Moreover, the production of polyunsaturated fatty acids from non-fatty acid substrates has not yet been shown in non-plant cells.
In WO 2004/057001 the inventors describe that the technology works in both plants and microorganims. However, the inventors have yet only confirmed the described technology in plants. PCT/US2004/014541 describes the production of PUFAs, such as arachidonic acid and EPA using oleaginous yeast. The inventors define oleaginous yeast as yeast that can accumulate at least 25% of its cell dry weight as oil. The invention uses oleaginous yeast such as Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces as host cell. It does not provide or claim information about other organisms or non-oleaginous yeast such as Saccharomyces cerevisiae. The inventors exemplify their technology by heterologous expression of three additional enzymes in Yarrowia lipolytica using delta-6 desaturase, delta-5 desaturase and delta-17 desaturase. The latter is equivalent to omega-3 desaturases. This approach makes use of the endogenous delta-12 desaturase.
In WO2005/01236 the inventors show that it is possible to produce PUFAs in yeast by supplying a fatty acid together with a non-fatty acid substrate. The inventors express delta-4 desaturase, elongases and/or delta-5 desaturase in Saccharomyces cerevisiae. By providing EPA or stearidonic acid together with galactose, Saccharomyces cerevisiae produces DHA.
PUFAs are increasingly supplied in food, for example in infant formula, and also in pharmaceutical formulations. A general source of PUFAs is fish oil. However, the fatty acid content of fish oil may vary during the fishing season and in some cases the fish oil may be contaminated because of environmental pollution. Besides this, fish oil has an obnoxious smell, which precludes its use as a food supplement.
Hence, proper and expensive purification steps are necessary for some application of PUFAs. The need for PUFAs produced by well-defined methods and in large quantities will increase dramatically during the next 5-10 years, and it is estimated that PUFAs will be used in many different products as a supplement. In order to meet the increasing demand for high quality PUFAs focus has moved towards reproducible production methods and this includes production methods using non-fatty acids substrates. The latter allows a more defined production of unsaturated fatty acids.
The present invention addresses this demand, and presents an efficient new, cost effective and alternative method for the high-level production of mono unsaturated fatty acids and particular PUFAs.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.